1. Technical Field
The present invention relates to priming or activation of β2 (beta2) family of integrins with various agents. The present invention further relates to treating various diseases and conditions that involve beta2 family of integrins.
2. Background Art
Integrins are non-covalently linked α/β heterodimeric receptors that mediate cell adhesion, migration and signaling. Together with their ligands, integrins play central roles in many processes including development, hemostasis, inflammation and immunity, and in pathologic conditions such as cancer invasion and cardiovascular disease. Key leukocyte functions, such as activation, migration, tissue recruitment and phagocytosis, are essential for their normal immune response to injury and infection and in various conditions, including inflammatory and autoimmune disorders [1, 2]. The β2 (b2) integrins, a sub-family of α/β heterodimeric integrin receptors have a common β-subunit (β2, CD18) but distinct α-subunits (CD11a, CD11b, CD11c and CD11d [3]) [4]. They regulate leukocyte functions, including via highly expressed integrins CD11a/CD18 (also known as LFA-1) and CD11b/CD18 (also known as Mac-1, CR3 and αMβ2) [2] that recognize a variety of ligands. For example, CD11b/CD18 recognizes >30 ligands, including the complement fragment iC3b, Fibrinogen, CD40L and ICAM-1 as ligands, among various others. CD11b/CD18 has been implicated in many inflammatory and autoimmune diseases. These include ischemia-reperfusion injury (including acute renal failure and atherosclerosis), Multiple Sclerosis (MS), tissue damage, transplantation, lupus, lupus nephritis, macular degeneration, glaucoma, stroke, neointimal thickening in response to vascular injury and the resolution of inflammatory processes [5-9]. For example, leukocyte infiltration and plaques of demyelination in the brain and spinal cord of patients are a hallmark of MS and CD11b/CD18 has been shown to play a key role in mediating leukocyte adhesion, migration and trafficking in MS and is a validated target for MS. Similarly, influx of inflammatory leukocytes potentiates anti-GBM nephritis, which is a model of rapidly progressive glomerulonephritis and lupus nephritis, and is characterized by proteinuria, leukocyte infiltration and glomerular crescent formation [10, 11]. Leukocytes play a critical role in the pathogenesis of anti-GBM nephritis, and their number correlates with the percentage of crescentic glomeruli. CD11b−/− animals show no proteinuria and strong protection of renal function [12], suggesting that agents targeting this integrin have a potential to treat this disease.
According to the American Cancer Society, worldwide, nearly 8 M people die from cancer every year. This number is expected to rise to 13.1 M deaths per year by the year 2030. There were 13.2 M new cases of cancer in the world in 2008, with an associated cost burden of $290 B, and these cases are expected to rise to 22.2 M by 2030, with a cost burden of $458 B. The developing world sees twice as many new cases of cancer as the developed world. Cancer is the second most common cause of death in the US; nearly 600,000 Americans are expected to die of cancer in 2013, almost 1,600 people per day, accounting for nearly 1 of every 4 deaths. About 1.6 M new cancer cases are expected to be diagnosed in 2013.
Breast cancer (BC) is the second most common cancer among women in the US; 1 in 8 women will have BC in their lifetime; BC is also a leading cause of cancer death among women of all races; ˜226,000 new cases of invasive BC in 2012; almost 40,000 women die from BC every year. Besides being female, age is the most important risk factor for BC. BC produces no symptoms when the tumor size is small and large tumors may become evident as a breast mass, but are also often painless. Breast pain is more likely to be caused by benign conditions and is not a common early symptom of BC.
Currently, surgical removal of part or whole breast is the most effective treatment for early-stage BC, in combination with radio- and chemo-therapy. Postmenopausal women with early stage breast cancer that tests positive for hormone receptors benefit from treatment with an aromatase inhibitor (e.g., letrozole, anastrozole, or exemestane) in addition to, or instead of, tamoxifen. For women whose cancer tests positive for HER2/neu, approved targeted therapies include trastuzumab (Herceptin) and, for advanced disease, lapatinib (Tykerb) and pertuzumab (Perjetal). The US Food and Drug Administration (FDA) revoked approval of bevacizumab (Avastin) for the treatment of metastatic breast cancer in 2011 because of evidence showing minimal benefit and some potentially dangerous side effects. Thus, additional therapeutics that are more effective and have fewer side effects are greatly needed. Furthermore, adjuvant therapeutics that can significantly reduce the dose of toxic chemo- and radio-therapeutic regimens in patients with BC are greatly needed.
Also, a majority of currently used anticancer therapies have significant cardiovascular safety concerns. Dose-dependent and progressive left ventricular (LV) dysfunction manifesting as symptomatic heart failure is well documented in patients receiving anthracyclines. In women with early breast cancer, particularly those >65 years of age, cardiovascular disease (CVD) is now the most common cause of death as indicated by Surveillance, Epidemiology, and End Results (SEER)-Medicare linked data. Additionally, these women are also at increased risk of CVD compared with age-matched women without a history of breast cancer. Paclitaxel is arrythmogenic cytotoxic drug and leads to bradycardia, with incidence rate with paclitaxel ranging from 0.5% to 5% (and 1.7% with docetaxel). While the main cardiotoxicity of taxanes is bradycardia, ischaemia has also been described. Importantly, clinical trials with the newer therapeutics, such as human epidermal growth factor receptor 2 (HER2)-directed monoclonal antibodies (i.e. trastuzumab) and other newer multi-targeted small-molecule inhibitors show that interfere with molecular pathways crucial to normal cardiac homeostasis, resulting in relatively high incidences of subclinical and overt cardiac toxicity. Even more significantly, while the cardiac toxicity with newer therapies may be reversible, the recovery of LV function after treatment cessation is uncertain at this time. Trastuzumab (Herceptin, a humanized monoclonal antibody against the HER2 tyrosine kinase receptor) shows the incidence of LVEF decrease or asymptomatic heart failure (HF) by ˜7%, but it can rise to 13% when trastuzumab is administered with concurrent paclitaxel and to 27% with concurrent anthracyclines. Thus, there is a great need for newer therapeutics for BC, which, in addition to being more efficacious, also lower the cardiovascular risk.
Inflammatory Leukocytes Recruited to Tumor Microenvironment are Targets for Cancer Therapy. Inflammation is directly linked to rumor growth and re-growth post treatment with surgery, anti-cancer agents and radiation. CD45+ leukocytes are significantly upregulated in naïve human breast tumors and after chemo-therapy. Myeloid cells (e.g.; neutrophils and macrophages) are among the cell types that are highly upregulated in the tumors, especially post treatment. In multiple animal models, reducing infiltration of myeloid cells leads to significant reduction in tumor burden, improves efficacy of cancer therapies and reduces BC metastasis. For example, it was recently shown that anti-CD11b antibodies enhance tumor response to radiation in models of squamous cell carcinoma.
Leukocytic β2 integrins also modulate tumor infiltration. For example, tumors also secrete inflammatory cytokines to recruit CD11b-expressing myeloid cells to facilitate neovascularization [13]. During cancer treatments, irradiated tumors recruit large numbers of specific leukocytes, such as bone marrow-derived CD11b-expressing myeloid cells expressing matrix metalloproteinase-9 (MMP-9), that restore tumor vasculature and allow tumor re-growth and recurrence [14]. Recent studies have shown that treatment with CD11b antagonists (anti-CD11b antibody) reduces CD11b-expressing myeloid cell infiltration and an enhancement of tumor response to radiation in mice [14], suggesting that agents targeting this integrin have a potential to be used as therapeutics in oncology.
Additionally, exposure to ionizing radiation (IR) causes injury in animals, eliciting an influx of inflammatory leukocytes that is partly responsible for early (acute) and late (chronic) injury and progressive functional impairment of multiple critical organs in mammals [15-20]. These include the hematopoietic system. The consequences of exposure to ionizing radiation (IR) are of major concern for patients that have, for example, undergone radiation therapy and individuals that are exposed to IR due to nuclear accident or attack. Moreover, exposure to sublethal IR also causes dose-dependent injury, including the hematological toxicity and also affects both the hematopoietic stem cell (HSC) numbers and their function (functional damage), including their capacity for long-term repopulation [21-26]. Therefore, blockage or reduction of inflammatory responses after radiation exposure could help mitigate early (acute) and late (chronic) effects of radiation in exposed patients.
Furthermore, acquired bone marrow failure (BMF) develops after an injury to the bone marrow (BM) by ionizing radiation (IR), chemotherapy drugs and antibiotics (e.g. busulfan and chloramphenicol), toxic chemicals (benzene, carbon tetrachloride), or viral infection (hepatitis, HIV, CMV, parvovirus). Another form of acquired BMF called aplastic anemia is an immune-mediated BMF that develops after lymphocyte infusion, and is characterized by an immune-mediated functional impairment of hematopoietic stem cells (HSCs). Functional damage in HSCs can over time lead to development of acquired BMF.
CD11b/CD18 is also expressed on short-term repopulating hematopoietic stem cells (HSCs) and hematopoietic progenitors (HPCs), and has been shown to participate in the retention and anchoring of HPCs in the bone marrow during enforced mobilization, suggesting that agents targeting CD11b/CD18 can have a protective effect on the number and function of HSCs and HPCs, in vitro, ex vivo, and in vivo.
Studies over the last several years have shown that blocking CD11b/CD18 and its ligands with antibodies and ligand mimics (anti-adhesion therapy) [24-26] and genetic ablation of CD11 b or CD18 decreases the severity of inflammatory response in vivo in many experimental models [27, 28]. However, such blocking agents have had little success in treating inflammatory/autoimmune diseases in humans [28, 29], perhaps because complete blockage of CD11b/CD18 with antibodies is difficult due to availability of a large mobilizable intracellular pool of CD11b/CD18 [30, 31] or because suppressing leukocyte recruitment with blocking agents requires occupancy of >90% of active integrin receptors [32]. Anti-integrin β2 antibodies have also shown unexpected side effects [33]. Additionally, whether transient activation of a fraction of native integrin receptors in vivo, as is expected from treatment with an activating agent, will have any significant biological effect in physiologically relevant settings remains an open question.
A number of published reports in the literature show that, in addition to increasing cell adhesion and modulating migration, CD11b/CD18 activation mediates a number of intracellular signaling events, mediate a number of intracellular signaling events, including production of reactive oxygen species and modulation of a number of pro- and anti-inflammatory genes in inflammatory cells [27-32]. Integrin activation and ligand binding leads to its clustering on the cell surface and initiates outside-in signaling, including the activation of PI3-K/Akt and MAPK/ERK1/2 pathways [28, 33], thereby mimicking the anchorage-dependent pro-survival signals in most cells. Ligation and clustering of integrins also synergistically potentiates intracellular signaling by other receptors (such as, Toll-like receptors (TLRs) and cytokine receptors interleukin-1 receptor (IL-1R) and TNFR) and both induce transcription factor (such as, NF-κB) dependent expression of pro-inflammatory cytokines (e.g.; IL1β, IL6, TNF-α) as well as release of other factors (e.g.; Tissue Factor). CD11b/CD18 deficiency enhances TLR4-triggered production of pro-inflammatory cytokines. The above suggests that CD11b/CD18 and its activation has a protective role in healthy mammals and that in inflammatory conditions or diseases, CD11b/CD18 activation would also suppress inflammation, inflammatory injury and disease by negatively regulating pro-inflammatory pathways in CD11b/CD18-expressing cells [34-36].
The above also suggests that there is a considerable potential for agents that modulate the function of CD11b/CD18 as therapeutic agents for the treatment of various inflammatory conditions. CD11b/CD18 is normally expressed in a constitutively inactive conformation in circulating leukocytes and in many other cells, but is rapidly activated to mediate its various biological functions [23]. CD11b/CD18 is also expressed on many cell types and tissues, including microgila, hepatocytes, HSCs, HPCs and a sub-type of T- and B-cells. CD11b/CD18 is also found in its cleaved, soluble form in some instances [37].
Blocking beta2 integrins, including CD11b/CD18, and their ligands with antibodies and ligand mimics (anti-adhesion therapy) [38-40] and genetic ablation of CD11a, CD11b, CD11c or CD18 decreases the severity of inflammatory response in vivo in many experimental models [41-43]. However, such blocking agents have had little success in treating inflammatory/autoimmune diseases in humans [42, 44], perhaps because complete blockage of integrins with antibodies is difficult due to availability of a large mobilizable intracellular pool of such integrins (for example, CD11b/CD18) [45, 46] or because suppressing leukocyte recruitment with blocking agents requires occupancy of >90% of active integrin receptors [47]. Anti-integrin 62 antibodies have also shown unexpected side effects [48]. Additionally, whether transient activation of a fraction of native integrin receptors in vivo, as is expected from treatment with an activating agent, will have any significant biological effect in physiologically relevant settings remains an open question.
Therefore, there is a considerable need for novel agents, such as antibodies, proteins, peptides, chemical compounds and small molecules, that selectively regulate the ligand binding and function of β2 integrins, including integrins CD11a/CD18, CD11b/CD18 and CD11c/CD18. Additionally, there is a need for agents that activate integrins (agonists). Such agonists can enhance the function of β2 integrins by, for example, targeting or binding to an allosteric regulatory site, such as the hydrophobic site-for-isoleucine (SILEN) pocket in CD11b/CD18, and other similar sites, but not the ligand-binding site on the integrin. Thus, there is a need for integrin activating agents that do not block ligand-binding functions of integrins. Moreover, agents and methods to enhance or promote integrin-mediated cell-adhesion and cellular functions are highly desired. However, progress towards identifying such agonists has been slow, especially agonists that selectively target and activate β2 integrins, including CD11b/CD18, with only a few reported discoveries [49, 50].
The present invention describes novel CD11b/CD18 agonists and a novel approach that involves integrin CD11b/CD18 priming for activation or activation, rather than its blockade, as a strategy for modulating CD11b/CD18 function. Such biological functions include cell adhesion, ligand binding, migration, phagocytosis, and the generation of effector molecules, such as cytokines. The present invention further describes compounds and approaches for modulating the function of CD11b/CD18 expressing cells (such as leukocytes, microglia, hepatocytes and lymphocytes), including their adhesion, migration, recruitment and other biological functions. It was strategized that various agents, such as small molecules, which are easily delivered in vivo and can be readily optimized for use in different mammals, would be the best approach for activating integrins. Here, it is shown that, without limitation, inflammatory disease can be reduced by CD11b/CD18 activation with novel small molecules. This shows that integrin activation is a novel, useful, pharmacologically targetable methodology to treat, without limitation, a variety of inflammatory and autoimmune diseases and conditions.
The present invention also shows that CD11b/CD18 activating agents that activate the normal wild type form of CD11b/CD18 and any of its mutant forms, such as the R77H mutant commonly found in many autoimmune disease carrying patients [51], would be highly beneficial. This invention describes a novel strategy, as an alternative to the anti-adhesion strategy that is currently practiced in literature, for regulating the biological function of integrins and integrin-expressing cells. Many different types of agents can activate integrins, such as biologics, antibodies, antibody fragments, proteins, lipids, oligonucleotides and chemical compounds.
An important requirement of useful agonists and compositions that regulate β2 integrins, including CD11b/CD18, is that they do not negatively impact the cell, tissue and animal viability. It is an object of the present invention to describe such agonists, compositions and methods. In addition, it is an objective of the present invention to show that transient activation of a fraction of native receptors in vivo, as is expected from treatment with an agonist and method of this invention, has a biological effect in physiologically relevant model systems. In addition, the present invention provides other related advantages. Moreover, an important requirement of useful compounds and compositions that regulate beta2 integrins, including CD11b/CD18, is that they not negatively impact the cell, tissue and animal viability. Some have suggested that integrin agonists might induce killing of target cells (Yang et al., J Biol Chem 281, 37904 (2006)), which is not desirable. Also, there is some prior art on the thiazolidine-one family of compounds, including U.S. Pat. Nos. 5,225,426, 7,566,732, 7,348,348, US 2006/0281798, US 2006/0183782, US 2006/0106077, US 2008/0108677, US 2010/0056503, WO 2009026346, WO/1995/029243. However, no compounds or methods with above described desirable properties have so far been described in the literature. It is an object of the invention to describe such compounds and methods. In addition, the present invention provides other related advantages.
Furthermore, integrins are now shown to exist in more than two conformations (closed and open). For example, CD11a/CD18 has been shown to exist in at least three conformations—closed, intermediate and open—based on its affinity for its ligand ICAM-1 in each of these states [52]. This also suggests, although has not been previously shown, that these different integrin conformations will induce different intracellular signaling pathways. It is an object of the current invention to describe β2 integrin agonists that, upon binding to β2 integrins, activate the β2 integrins and induce intracellular signaling pathways that are different from the ligand-bound β2 integrin conformation(s).
Moreover, a number of agents currently under development as anti-inflammatory agents are targeted towards specific kinases, such as spleen tyrosine kinase (Syk), T cell receptor-associated protein kinase (ZAP70), Janus kinases (JAKs) and Bruton's tyrosine kinase (BTK) [53]. There remains a need for compounds and methods that effectively treat inflammation, especially targeting those kinases.